Compositions and methods to modulate immune and inflammatory responses

ABSTRACT

The present invention is directed to compositions and methods to modulate inflammatory and immune responses in a subject, which includes immune activation and inhibiting inflammatory responses, by modulating the regeneration and tolerance factor (RTF).

BACKGROUND OF THE INVENTION

[0001] The inflammatory process initiates a localized defense againstforeign antigens. The process is characterized by immune activation aswell as destruction of nearby tissues secondary to involved release ofdestructive agents. Initially involved tissue assumes a significant rolein provoking the inflammatory response at large (Matzinger, P., Thedanger model: a renewed sense of self. Science, 2002. 296(5566): p.301-5; Medzhitov, R. and C. A. Janeway, Jr., Decoding the patterns ofself and nonself by the innate immune system. Science, 2002. 296(5566):p. 298-300). One of the primary immunogenic mechanisms triggered by theinflammatory response includes the release of ATP into the extracellularspace. ATP is liberated from injured tissue itself—as well as fromnearby macrophages, neutrophils, platelets and dying cells—followingincidents of stress, infection or other forms of tissue insult(Fredholm, B. B., Purines and neutrophil leukocytes. Gen Pharmacol,1997. 28(3): p. 345-50; Beigi, R., et al., Detection of local ATPrelease from activated platelets using cell surface-attached fireflyluciferase. Am J Physiol, 1999. 276(1 Pt 1): p. C267-78; Schwiebert, E.M., ABC transporter-facilitated ATP conductive transport. Am J Physiol,1999. 276(1 Pt 1): p. C₁₋₈; Mizumoto, N., et al., CD39 is the dominantLangerhans cell-associated ecto-NTPDase: modulatory roles ininflammation and immune responsiveness. Nat Med, 2002. 8(4): p. 358-65;Dubyak, G. R. and C. el-Moatassim, Signal transduction via P2-purinergicreceptors for extracellular ATP and other nucleotides. Am J Physiol,1993. 265(3 Pt 1): p. C577-606; Gordon, J. L., Extracellular ATP:effects, sources and fate. Biochem J, 1986. 233(2): p. 309-19).Extracellular ATP can then exert a wide variety of effects onsurrounding cells, most notably the induction of apoptotic eventsleading ultimately to cell death in both lymphocytes and macrophages (DiVirgilio, F., The P2Z purinoceptor: an intriguing role in immunity,inflammation and cell death. Immunol Today, 1995. 16(11): p. 524-8;Gargett, C. E., J. E. Cornish, and J. S. Wiley, ATP, a partial agonistfor the P2Z receptor of human lymphocytes. Br J Pharmacol, 1997. 122(5):p. 911-7; Buisman, H. P., et al., Extracellular ATP induces a largenonselective conductance in macrophage plasma membranes. Proc Natl AcadSci USA, 1988. 85(21): p. 7988-92; Ferrari, D., et al., ATP-mediatedcytotoxicity in microglial cells. Neuropharmacology, 1997. 36(9): p.1295-301; Zheng, L. M., et al., Extracellular ATP as a trigger forapoptosis or programmed cell death. J Cell Biol, 1991. 112(2): p.279-88). Diminution of cell integrity then leads to further ATP release,propagating the inflammatory response and continued tissue destruction.Recent data have shown an attenuated effect of extracellular ATP in thepresence of the regeneration and tolerance factor (RTF) protein. Thepresent invention discloses compositions and methods to modulateinflammatory and immune responses by regulating the activities of RTF.RTF prevents ATP activation of P2Z, thus preventing the cell fromundergoing pro-inflammatory activation and apoptosis.

SUMMARY OF THE INVENTION

[0002] The present invention is directed to compositions and methods tomodulate inflammatory and immune responses in a subject, which includesimmune activation and inhibiting inflammatory responses, by modulatingthe regeneration and tolerance factor (RTF). Immune activation can beaccomplished by inhibiting the RTF activity. In one embodiment, the RTFactivity can be inhibited by administering a RTF antagonist, which canbe a small molecule, a peptide, or an antibody or a fragment thereof.Inhibiting the RTF activity may result in cell apotosis, especiallycancer cells such as ovarian carcinoma cells.

[0003] Inflammatory responses can be inhibited by increasing the levelof activity of RTF. In an embodiment, the method of increasing the levelof activity of RTF is by administering an effective amount of RTF or afragment of RTF. The RTF or its fragment can be isolated or purifiedfrom mammalian cells such as the T-lymphocyte, the B-lymphocyte, themacrophage, thymus, or the fetalplacental tissue. Alternatively, the RTFcan be synthetic or a recombinant RTF.

[0004] The present invention is also directed to a method of treatingcancer, such as ovarian cancer, by inhibiting the RTF activity. In oneembodiment, the method to inhibit RTF activity is to administer aneffective dose of a RTF antagonist.

[0005] The present invention is further directed to a method of treatingor ameliorating an inflammatory disorder or an autoimmune disorder orone or more symptoms thereof by administering an effective amount of RTFor a fragment of RTF. The RTF or its fragment can be isolated orpurified from mammalian cells such as the T-lymphocyte, theB-lymphocyte, the macrophage, thymus, or the fetalplacental tissue.Alternatively, the RTF can be synthetic or a recombinant RTF. The RTFcan be formulated with one or more suitable pharmaceutical excipientsfor administration by parenteral, intramuscular, topical, nasal,pulmonary, or oral route. The formulation can also be in the form ofmicroparticles or nanoparticles. In an embodiment, the method oftreating or ameliorating an inflammatory disorder or an autoimmunedisorder or one or more symptoms thereof includes an additional step ofadministering a TNF-α antagonist, which includes but is not limited toinfliximab, etanercept, D2E7, CDP571, CDP870, thalidomide and itsanalogs, and phosphodiesterase type IV inhibitors.

[0006] Additional features and advantages of the present invention aredescribed in, and will be apparent from, the following DetailedDescription of the Invention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1 shows the expression of RTF and P2Z on PBMC andmacrophages. Flow cytometric analysis of the expression of RTF and P2Z.Dotted line indicates isotype control antibody. Solid line representseither RTF expression (column 1) or P2Z expression (column 2). The firstrow represents unstimulated PBMC. The second row represents PBMCstimulated for 72 hours with PHA. The third row represents J774A1macrophages.

[0008]FIG. 2 illustrates that RTF expression is concurrent with P2Zexpression on lymphocytes during stimulation. Lymphocytes werestimulated with PHA for 0-72 hours. The expression on RTF and P2Z wasmeasured at time points 0, 24 hrs, and 72 hrs as indicated by flowcytometry. The X axis represent the percent maximal expression of eachmolecule. The black bars represent RTF expression and the gray barsrepresent P2Z expression. Error bars represent the standard error of themean.

[0009]FIG. 3 shows that RTF moves to the surface of the membrane in a 50kDa form during activation. PBMC were stimulated for 0-72 hours and RTFsize was examined by western blot and RTF location was examined byconfocal microscopy. Panel A: Western Blot: PBMC were stimulated withplate bound CD3 and CD28 and harvested at the time points indicated (inhours), western blotted and probed by 2C1. Row 1 represents proteins of70 kDa size, and row 2 represents proteins of 50 kDa size. Each lanerepresents a stimulation time of 0, 18, 48, and 72 hours respectively.Panel B: Confocal Microscopy: Confocal microscopy images of PBMC takenbefore activation (left panel) and at 72 hrs of activation (rightpanel), permeabilized and stained with anti-RTF-FITC and counterstainedwith anti-CD8-PE.

[0010]FIG. 4 demonstrates that surface ATPase activity is inhibited byanti-RTF. PBMC and J774A1 macrophages with anti-RTF antibody (blackcolumns) or isotype control antibody (gray columns) were examined forsurface ATPase activity, by adding [32]γP-ATP and measuring the releaseof [32]-Pi. The Y axis represents surface ATPase activity as measured incounts per minute [32]-Pi. Error bars represent the standard error ofthe mean.

[0011]FIG. 5 shows that anti-RTF and ATP generate more apoptosistogether than either alone. Top row represent flow cytometry histogramsof J774A1 macrophages incubated with ATP, anti-RTF, or both together.Bottom row represents J774A1 cells incubated with ATP, anti-RTF, anddifferent concentrations of ATPase as indicated. X axis representsannexin-V staining. Means of four independent experiments with standarderror are given for each.

[0012]FIG. 6 demonstrates the enhancement of secretion of IL-1β in THP-1Macrophages by anti-RTF antibody.

[0013]FIG. 7 illustrates the cytotoxic effect of anti-RTF antibody toovarian carcinoma cells.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention is directed to compositions and methods tomodulate inflammatory and immune responses in a subject by regulatingthe level of activity of regeneration and tolerance factor (RTF).Modulating inflammatory and immune responses includes both immuneactivation and the inhibition of the inflammatory responses. In apreferred embodiment, the subject is a mammal. In a more preferredembodiment, the subject is a human.

[0015] A principle means by which RTF controls the extent ofinflammation is via its interaction with P2Z, a cell surface receptorexpressed primarily on cells of hematopoetic origin such as lymphocytesand macrophages. P2Z is one of a family of receptors known aspurinoceptors which are activated by nucleotides. Extracellular ATPbinds P2Z, which triggers the opening of specific calcium channels aswell as non-specific transmembrane channels (Dubyak, G. R. and C.el-Moatassim, Signal transduction via P2-purinergic receptors forextracellular ATP and other nucleotides. Am J Physiol, 1993. 265(3 Pt1): p. C577-606; Gargett, C. E., J. E. Cornish, and J. S. Wiley, ATP, apartial agonist for the P2Z receptor of human lymphocytes. Br JPharmacol, 1997. 122(5): p. 911-7; Zheng, L. M., et al., ExtracellularATP as a trigger for apoptosis or programmed cell death. J Cell Biol,1991. 112(2): p. 279-88). These channels then allow for rapid influx ofcalcium ions into the cytosolic space. This calcium influx in turndrives forward many specific activation pathways. The activation of P2Zcan cause specific activation of NF-κB, and the maturation and secretionof IL-1β, a potent inflammatory cytokine. This will subsequentlyincrease both cellular activation and drive the inflammatory response,making P2Z a key mediator in the inflammatory process. It can also causeapoptosis and an inflammatory counterpart to apoptosis, oncosis,inducing the release of further ATP. Recent reports have shown that theabsence of the P2Z purinoceptor can attenuate the inflammatory response(Labasi J M, P. N., Donovan C, McCurdy S, Lira P, Payette M M, BrissetteW, Wicks J R, Audoly L, Gabel C A., Absence of the P2X7 receptor altersleukocyte function and attenuates an inflammatory response. J Immunol,2002. 168(12)(Jun 15): p. 6436-45).

[0016] Therefore it follows that extracellular ATP up-regulates theinflammatory response through its instigation of P2Z activation at thesite of inflammation and also the stimulated release of pro-inflammatorycytokines. A key regulatory point at which to reduce the inflammatoryresponse, then, is in the concentration of extracellular ATP. It hasbeen suggested that ecto-ATPases hold important regulatory surfaceATPase activity, which attenuates inflammatory as well as immuneresponses (Labasi J M, P. N., Donovan C, McCurdy S, Lira P, Payette M M,Brissette W, Wicks J R, Audoly L, Gabel C A., Absence of the P2X7receptor alters leukocyte function and attenuates an inflammatoryresponse. J Immunol, 2002. 168(12)(Jun 15): p. 6436-45; Filippini, A.,et al., Ecto-ATPase activity in cytolytic T-lymphocytes. Protection fromthe cytolytic effects of extracellular ATP. J Biol Chem, 1990. 265(1):p. 334-40). The present application discloses that RTF is the primarysurface ATPase with this function.

[0017] RTF is the α-subunit of α2-isoform of vacuolar ATPase, and thatRTF exists as a 70 kDa protein (Toyomura, T., et al., Three subunit aisoforms of mouse vacuolar H(+)-ATPase. Preferential expression of thea3 isoform during osteoclast differentiation. J Biol Chem, 2000.275(12): p. 8760-5; Nichols, T. C., et al., Expression of a membraneform of the pregnancy-associated protein TJ6 on lymphocytes. CellImmunol, 1994. 155(1): p. 219-29; Boomer, J. S., et al., Regenerationand tolerance factor's potential role in T-cell activation andapoptosis. Hum Immunol, 2000. 61(10): p. 959-71). A 20 kDa fragment canbe cleaved, leaving the 50 kDa external membrane form (Nichols, T. C.,et al., Expression of a membrane form of the pregnancy-associatedprotein TJ6 on lymphocytes. Cell Immunol, 1994. 155(1): p. 219-29).Current data support that RTF is known to have a role in certain formsof apoptosis (Boomer, J. S., et al., Regeneration and tolerance factor'spotential role in T-cell activation and apoptosis. Hum Immunol, 2000.61(10): p. 959-71; Boomer, J. S., et al., Regeneration and tolerancefactor is expressed during T-lymphocyte activation and plays a role inapoptosis. Hum Immunol, 2001. 62(6): p. 577-88). It has also been shownequally that anti-RTF antibody can block RTF's ATPase activity,subsequently allowing ATP to bind P2Z and induce apoptosis. It hasadditionally been shown that RTF works in an antagonistic fashion versusthe P2Z surface purinoceptor. U.S. Pat. No. 6,133,434 and InternationalPatent Application (PCT) No. WO 95/33048 disclose that antibodiesagainst P2Z receptors can be useful in treating a variety of diseasesand conditions, including epilepsy, cognition, emesis, pain (especiallymigraine), asthma, peripheral vascular disease, hypertension, diseasesof immune system, irritable bowel syndrome and premature ejaculation.

[0018] One possible mechanism for RTF to modulate inflammatory andimmune responses is by RTF acting as an ATPase to reduce the level ofextracellular ATP. ATP, released during injury or stress from tissues,infiltrating cells, and dying cells at the site of inflammation,interacts with the P2Z receptor. This interaction exacerbates theinflammatory response which leads to cell activation and the maturationand secretion of IL-1β. By controlling the degradation of extracellularATP, RTF down regulates immune responses, including immune activationand inflammation. Thus, inhibiting the RTF activity leads to immuneactivation, which may lead to cell death, while enhancing the RTFactivity leads to inhibition of inflammatory responses.

[0019] Any method that inhibits RTF activity can be employed in thepresent invention for immune activation. In a preferred embodiment, themethod to inhibit RTF is by administering a RTF antagonist. The RTFantagonist can be a small molecule, a peptide, an antibody or a fragmentthereof, and the like. The antibody can be either polyclonal ormonoclonal. In one embodiment, the antagonist acts by binding to RTF toinhibit its activity. A preferred RTF antagonist is an anti-RTF antibodyor a fragment thereof, and a preferred anti-RTF antibody is a monoclonalRTF antibody or a fragment thereof. A method to produce and purify amonoclonal RTF antibody is disclosed by Boomer et al. (Boomer, J. S., etal., Regeneration and tolerance factor's potential role in T-cellactivation and apoptosis. Hum Immunol, 2000. 61(10): p. 959-71), whichis incorporated herein by reference and made a part hereof. In anotherembodiment, the antagonist acts by inhibiting cellular expression ofRTF. An example of such an antagonist includes, but is not limited to,an antisense nucleic acid which binds to the DNA coding for the RTFmolecule. Using antisense nucleic acid to bind to DNA to inhibitcellular expression of proteins is well known in the art. Anotherexample of antagonizing RTF is by inducing RNA interference (RNAi). TheRNAi process induces the degradation of the target gene mRNA so as tosilence the target gene expression. A typical RNAi process includesintroducing into cells a double-stranded interfering RNA that comprisesa sense RNA having the sequence homologous to the target gene mRNA andantisense RNA having the sequence complementary to the sense RNA. Thedouble-stranded interfering RNA can be a small interfering doublestranded RNA (also known as siRNA). The double-stranded interfering RNAcan be introduced to the cells exogenously, or it can be expressedintracellular by incorporating into the cells an expression system toexpress the double-stranded interfering RNA. Intracellular expression ispreferred for siRNA.

[0020] The RTF antagonist can be prepared as a pharmaceuticalformulation by including one or more suitable or appropriate excipients.In an embodiment, the pharmaceutical formulation is a nanoparticle or amicroparticle. The antagonist can be administered to the subject by anyroute including but not limited to parenteral, topical, nasal,pulmonary, ophthalmic, bucal, vaginal, transdermal, intrathecal, andoral

[0021] One of the results of inhibiting RTF activity is induction ofcell apotosis, especially cancer cells, such as ovarian carcinoma cells.Thus, inhibiting RTF can be used as a method to treat cancer. Any of themethods described above which inhibit RTF can then be used as a methodto treat cancer. An example of cancer that can be treated in the presentinvention is ovarian cancer.

[0022] Since inflammatory responses are the result of immune activationby the injured tissue, it is anticipated that enhancing RTF activitywill result in inhibiting the inflammatory responses. Inflammation isknown in many disorders which include, but are not limited to, arthritis(including but not limited to rheumatoid arthritis,spondyloarthopathies, gouty arthritis, osteoarthritis, systemic lupuserythematosus and juvenile arthritis), asthma, bronchitis, menstrualcramps, tendonitis, bursitis, and skin related conditions (such aspsoriasis, eczema, burns and dermatitis), certain gastrointestinalconditions (such as inflammatory bowel disease, Crohn's disease,gastritis, irritable bowel syndrome and ulcerative colitis), vasculardiseases, migraine headaches, periarteritis nodosa, thyroiditis,aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, type Idiabetes, myasthenia gravis, multiple sclerosis, sarcoidosis, nephroticsyndrome, Behcet's syndrome, polymyositis, gingivitis, hypersensitivity,conjunctivitis, swelling occurring after injury, myocardial ischemia,certain central nervous system disorders (such as Alzheimer's diseaseand dementia), allergic rhinitis, respiratory distress syndrome,endotoxin shock syndrome, atherosclerosis, central nervous system damageresulting from stroke, ischemia, trauma, and the like. Many of the aboveinflammatory disorders are autoimmune disorders in which the bodydevelops immune responses to its own protein. Thus, enhancing theactivity of RTF can be used to treat or prevent inflammatory disorders,including autoimmune disorders, or their symptoms.

[0023] Any method that enhances the level of activity of RTF can be usedto inhibit inflammatory responses and to treat or prevent inflammationin the present invention. In a preferred embodiment, the method is toadminister to the subject an effective amount of RTF or a fragmentthereof. In an embodiment, the RTF is an intracellular form which has amolecular weight of about 70 kDa. In another embodiment, the RTF isexternal membrane form having a molecule weight of about 50 kDa.

[0024] In one embodiment, the RTF can be isolated or purified from amammalian source, such as murine or human, which includes but is notlimited to, lymphocyte, macrophage, thymus, fetalplacental tissue, andthe like. The lymphocyte can be a T-lymphocyte or a B-lymphocyte. The T-or B-lymphocyte preferably is activated.

[0025] In another embodiment, the RTF or a fragment thereof can besynthetic or a recombinant RTF, which can be derived, for example, froma nucleic acid sequence (e.g. cDNA) encoding the amino acid sequence ofthe RTF or the fragment. The nucleic acid sequence can be incorporatedby recombinant techniques into an appropriate host organism for theexpression of the RTF or the fragment. Examples of host organismsinclude, but are not limited to, bacteria, fungi, yeasts, protozoa,transgenic animals, transgenic plants, and the like. Recombinanttechniques are well known to those skilled in the art and manybiopharmaceuticals have been successfully produced by such techniques. Amethod for producing a recombinant RTF in E. coli is disclosed by Lee etal. (Lee, G. W., Bommer, J. S., Gilman-Sachs, A., Chedid A., Gudelj, L.,Rukavina, D. and Beaman, K. D. Regeneration and tolerance factor of thehuman placenta induces IL-10 induction. Eur J Immunol, 2001. 31: p.687-691) which is incorporated herein by reference and made a parthereof.

[0026] In a preferred embodiment of the present invention, the RTF orthe fragment is formulated with one or more acceptable pharmaceuticalexcipients for administration to the subject. The RTF or the fragmentcan also be formulated as microparticles or nanoparticles. Themicroparticles or nanoparticles can further include ligands to deliverthe microparticles or the nanoparticles to a target tissue such as theT- or B-lymphocyte or the macrophage. The RTF or the fragment can beadministered to the subject by any acceptable route including, but notlimited to, parenteral, topical, nasal, pulmonary, ophthalmic, bucal,vaginal, transdermal, intrathecal, oral and the like.

[0027] In another embodiment, the method to enhance RTF activity can bea method which enhances the expression of cellular RTF.

[0028] In yet another embodiment of the present invention, the method oftreating inflammation by enhancing the RTF activity may include anadditional step of administering to the subject a TNF-α antagonist.Since both IL-1β and TNF-α are known to cause inflammation, inhibitingthe secretion of IL-1β by RTF and blocking the TNF-α activity by a TNF-αantagonist should have synergistic effects in treating inflammation.There are many known TNF-α antagonists. Examples include, but are notlimited to, infliximab, etanercept, D2E7, CDP571, CDP870, thalidomideand its analogs, and phosphodiesterase type IV inhibitors.

EXAMPLES Example 1 Cell Culture

[0029] J774A1 cells were maintained by allowing them to adhere to thebottom of a T75 flask with 25 mls of RPMI 1640 complete media at 37° C.in a 5% CO₂ atmosphere. Cells were split into a new flask every 4 daysusing trypsin and a gentle wash with RPMI.

Example 2 ATPase Assay

[0030] PBMC or J774A1 macrophages were suspended in 50 μl of RPMI at adensity of 2.5×10⁵ in a 96 well flat bottom plate and incubated with 10μg of either 2C1 (anti-RTF) or an isotype matched control antibody for45 mins at 37° C. in a 5% CO₂ atmosphere. Cells were washed with PBS andre-suspended in 100 μl of assay buffer (5 mM KCl, 2 mM CaCl₂, 2 mMMgCl₂, and 1.5 μC of [γ-32P]ATP) in 1.5 ml eppendorf tubes. The ATPasereaction was allowed to continue for 10 mins at room temperature. 500 μlof 20% w/v activated charcoal (in distilled water) was added to scavengeany unused [γ-32P]ATP and allowed to sit for 10 mins on ice. The cellswere centrifuged at 3200×g for 5 mins, pelleting the charcoal. 500 μl ofsupernatant containing released γ-32P was removed and added toscintillation vials with 3 mls of scintillation fluid and read in thescintillation counter for 1 min/tube (Filippini, A., et al., Ecto-ATPaseactivity in cytolytic T-lymphocytes. Protection from the cytolyticeffects of extracellular ATP. J Biol Chem, 1990. 265(1): p. 334-40;Dombrowski, K. E., et al., Identification and partial characterizationof an ectoATPase expressed by human natural killer cells. Biochemistry,1993. 32(26): p. 6515-22; Dombrowski, K. E., et al., Antigen recognitionby CTL is dependent upon ectoATPase activity. J Immunol, 1995. 154(12):p. 6227-37).

Example 3 Apoptosis Induction

[0031] Approximately 2.5×10⁵ PBMC were suspended in 100 μl of RPMI in a96 well round bottom plate and a final concentration of either 3 mM ATP,0.75 mM ATP, 10 μg anti-RTF, or 0.75 mM ATP+10 μg anti-RTF together wasadded. Cells were incubated in these conditions at 37° C. in 5% CO₂ for6 hours. Cells were washed twice in 0.01% BSA/PBS, and re-suspended in100 μl binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂)with 5 μl of annexin-V/FITC (BD/Phanningen) for 15 mins. Cells werewashed in binding buffer and re-suspended in 300 μl of binding bufferwith 3 μl of 1 mg/ml propidium iodide. Apoptosis was measured byquantifying annexin-V binding by flow cytometry by gating onPI-/annexin-V+ cells (Vermes, I., et al., A novel assay for apoptosis.Flow cytometric detection of phosphatidylserine expression on earlyapoptotic cells using fluorescein labeled Annexin V. J Immunol Methods,1995. 184(1): p.39-51).

Example 4 P2Z and RTF Staining

[0032] A total of 2.5×10⁵ cells were washed twice with 0.01% BSA/PBS for5 mins and re-suspended in 100 μl of staining buffer. 5 μl of rabbitanti-P2X₇ (P2Z) (Chemicon) antibody, 5 μl anti-RTF-FITC, or 5 μl ofisotype matched control antibody was added to the appropriate tubes andallowed in react in the dark for 15 mins. Cell were washed again andre-suspended in 100 μl of staining buffer (1×PBS, 5% FBS) with 5 μl ofanti-Rabbit biotinylated antibody (Pharmingen) and allowed to react for15 mins. A third wash was performed and 2 μl of Streptavidin-PE(Pharmingen) was added for 15 mins. After a final wash, the cells werere-suspended in 300 μl of staining buffer and run on a BD Facscaliburflow cytometer.

Example 5 Propidium Iodide and Lucifer Yellow Incorporation

[0033] J774 cells were suspended in 100 μl of RPMI in a 96 well roundbottom plate at a density of 2.5×10⁵ in on of the following conditions:3 mM ATP, 0.75 mM ATP, 50 μg anti-RTF, or 0.75 mM ATP+10 μg anti-RTF.Cells were incubated at 37° C. in 5% CO₂ for 30 mins in the presence of1 μg PI or LY, washed in PBS, re-suspended in 300 μl PBS and analyzed inthe BD Facscalibur flow cytometer.

[0034] For slides, J774 cells were grown in 4 well chamber slides for 2days. The supernatant was removed and each chamber was incubated in oneof the conditions described above for 15 mins in the presence of 1 μg PIor LY. The slides were washed in PBS, and incubated with 10μ Trypan bluefor 5 mins, washed again and examined under a fluorescent microscope.

Example 6 Confocal Microscopy

[0035] PBMC were obtained by ficoll separation and 2.5×10⁵ cells perwell were incubated in a 96 well plate in RPMI 1640 medium in thepresence of 2 μg/ml PHA, or RPMI alone. After 72 hours bothunstimulated, and stimulated cells were washed twice in PBS,permeabilized with ice cold 70% ethanol for 15 mins, and reacted with 7μl anti-RTF-FITC, anti-CD8-PE (Pharmingen), or isotype control antibodyfor 15 mins. Cells were washed again, fixed with Coulter fixativesolution, and a cytospin was used to transfer them to a positivelycharged slide which was mounted with Dapco medium. Slides were thenexamined by confocal microscopy.

Example 7 RTF Expression Follows P2Z Expression on Lymphocytes

[0036] It has been previously shown that RTF expression in lymphocytesis dependent on activation (Boomer, J. S., et al., Regeneration andtolerance factor's potential role in T-cell activation and apoptosis.Hum Immunol, 2000. 61(10): p. 959-7). To characterize RTF expression inrelation to P2Z expression during the immune response, the surfaceexpression of RTF and P2Z on lymphocytes was measured followingstimulation with PHA for 24-72 hours. In resting lymphocytes, RTFexpression was 14.42±0.06% that of maximal expression while P2Zexpression was 35.23±8.5%. RTF and P2Z expression increased together onthe surface of the cells as stimulation time increased, with RTF and P2Zexpression at 88.64±8.8% and 81.34±13.2% respectively at 72 hours (FIG.1). Both molecules matched the others percent maximal expression at 0and 24 hours, reaching maximal expression at 72 hours (FIG. 2).

[0037] The expression of RTF and P2Z on the J774A1 macrophage cell linehas also been examined. FIG. 1 shows that each molecule to be expressedin a constitutive manner.

Example 8 RTF Exists as a 50 kDa Protein on the Surface of ActivatedCells, and as a 70 kDa Protein Inside of Resting Cells

[0038] When lymphocytes examined by western blot at the time pointsstimulated above (FIG. 3a), unstimulated lymphocytes expressed a 70 kDaprotein of RTF as their major form of RTF. Upon stimulation, expressionof RTF fell below detectable levels at a time period of 16-24 hours. Ata time period of 24-72 hours after stimulation RTF expression returnedto detectable levels, however predominantly in a 50 kDa sized form (FIG.3a).

[0039] It is hypothesized that the 70 kDa form represents theintracellular form of RTF, while the 50 kDa form represents the externalmembrane form. To test this, the activated lymphocytes from FIG. 3a werefurther examined by confocal microscopy at 0 and 72 hours of stimulation(FIG. 3b). In this figure RTF (green) is present in the cytoplasm ofun-stimulated cells. In cells stimulated with PHA for 72 hrs, RTF hasre-localized to the surface of the membrane. Consistent with the westernblot shown in FIG. 3a, these findings suggest to us that the 70 kDaprotein is intracellular in resting lymphocytes, whereas the 50 kDaprotein is found on the surface of activated lymphocytes.

Example 9 RTF Regulates Surface ATPase Activity in Lymphocytes andMacrophages

[0040] It is hypothesized that RTF regulates surface ATPase activity andanti-RTF blocks it. To demonstrate that RTF has surface ATPase activity,PBMC and J774A1 macrophages were incubated with either anti-RTF orisotype control antibody for 45 mins, and their surface ATPase activitywas measured by [γ32-P]ATP degradation (Filippini, A., et al.,Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from thecytolytic effects of extracellular ATP. J Biol Chem, 1990. 265(1): p.334-40; Dombrowski, K. E., et al., Identification and partialcharacterization of an ectoATPase expressed by human natural killercells. Biochemistry, 1993. 32(26): p. 6515-22; Dombrowski, K. E., etal., Antigen recognition by CTL is dependent upon ectoATPase activity. JImmunol, 1995.154(12): p.6227-37). The PBMC incubated with anti-RTF hada 10 fold decrease in surface ATPase activity with 11,758±3,394 cpmcompared to PBMC incubated with isotype antibody with a cpm of115,592±23,891. (P<0.001; FIG. 4). The J774A1 cells incubated withanti-RTF had a complete inhibition of surface ATPase activity comparedwith the isotype control (45,752±16,175 cpm; P<0.001; FIG. 4). Thinlayer chromatography confirmed that anti-RTF completely inhibited thesurface ATPase activity (data not shown).

Example 10 RTF Prevents ATP from Inducing Apoptosis

[0041] Since RTF moves to the surface of the membrane as a 50 kDa sizeprotein after stimulation in PBMC, and RTF regulates ATPase activity, itis desirable to demonstrate that RTF can prevent ATP induced apoptosis.For this purpose the macrophage cell line J774A1 is chosen. J774A1 cellswere to be used as a model to demonstrate the interaction of RTF withP2Z because this cell line expresses both molecules constitutively andis known to undergo P2Z mediated, ATP induced apoptosis (Di Virgilio,F., The P2Z purinoceptor: an intriguing role in immunity, inflammationand cell death. Immunol Today, 1995. 16(11): p. 524-8; Coutinho-Silva,R. and P. M. Persechini, P2Z purinoceptor-associated pores induced byextracellular ATP in macrophages and J774 cells. Am J Physiol, 1997.273(6 Pt 1): p. C1793-800). If the theory that RTF regulates surfaceATPase activity and therefore regulates ATP binding to P2Z is correct,then it follows that anti-RTF and ATP would work together to generatemore apoptosis than either alone by blocking the ATPase activity of RTF.When added together, at the same concentration as both alone, a nearmaximal amount of apoptosis occurred as measured by annexin-V (FIG. 5).When 3 mM ATP was added, 84.14±1.63% of the cells underwent apoptosis,but when the amount of ATP was decreased to 0.75 mM, only 13.26±3.6%apoptosis was seen. When 10 μg of anti-RTF was added alone, 15.49±4.05%of the cells were apoptotic. However, anti-RTF and 0.75 mM ATP wereadded together, apoptosis was 83.05±5.09%. When ATPase was added back,no apoptosis occurred showing that RTF's apoptosis inducing ability isdependent on the presence of ATP (FIG. 5). Previously, in initialexperiments we demonstrated that anti-RTF had no effect on the ATPaseused, negating the possibility of their interaction (data not shown).

Example 11 RTF Prevents P2Z Activation

[0042] A standard technique for detecting P2Z activity is used todemonstrate that the ultimate cause of the apoptosis inducing effect ofRTF and ATP was due to P2Z activation (Di Virgilio, F., The P2Zpurinoceptor: an intriguing role in immunity, inflammation and celldeath. Immunol Today, 1995. 16(11): p. 524-8; Coutinho-Silva, R. and P.M. Persechini, P2Z purinoceptor-associated pores induced byextracellular ATP in macrophages and J774 cells. Am J Physiol, 1997.273(6 Pt 1): p. C1793-800; Steinberg, T. H., et al., ATP4-permeabilizesthe plasma membrane of mouse macrophages tofluorescent dyes. J BiolChem, 1987. 262(18): p. 8884-8). When ATP interacts with P2Z,non-specific channels of about 0.9 kDa in size will open in the membrane(Dubyak, G. R. and C. el-Moatassim, Signal transduction viaP2-purinergic receptors for extracellular ATP and other nucleotides. AmJ Physiol, 1993. 265(3 Pt 1): p. C577-606; Di Virgilio, F., The P2Zpurinoceptor: an intriguing role in immunity, inflammation and celldeath. Immunol Today, 1995. 16(11): p. 524-8; Hickman, S. E., et al.,P2Z adenosine triphosphate receptor activity in cultured humanmonocyte-derived macrophages. Blood, 1994. 84(8): p. 2452-6; Greenberg,S., et al., Extracellular nucleotides mediate Ca2+ fluxes in J774macrophages by two distinct mechanisms. J Biol Chem, 1988. 263(21): p.10337-43). This allows the testing for P2Z's influence by measuring theinflux of molecules smaller than 0.9 kDa, to which the cell is normallynot permeable. Propidium iodide (PI) (0.414 kDa) and lucifer yellow (LY)(0.46 kDa) should be allowed through if P2Z is activated, and trypanblue (TB) (0.96 kDa) should be excluded under all circumstances unlessnecrosis has occurred (Di Virgilio, F., The P2Z purinoceptor: anintriguing role in immunity, inflammation and cell death. Immunol Today,1995. 16(11): p. 524-8; Coutinho-Silva, R. and P. M. Persechini, P2Zpurinoceptor-associated pores induced by extracellular ATP inmacrophages and J774 cells. Am J Physiol, 1997. 273(6 Pt 1): p.C1793-800; Steinberg, T. H., et al., ATP4-permeabilizes the plasmamembrane of mouse macrophages tofluorescent dyes. J Biol Chem, 1987.262(18): p. 8884-8). The same conditions used in the previous experimentwere repeated to observe and measure the entry of propidium iodide ortrypan blue in J774A1 cells. Thus P2Z influence would be characterizedby cells that were PI and LY positive but TB negative.

[0043] Using flow cytometry, cells incubated with anti-RTF+0.75 mM ATPhad pronounced channel formation judging from their incorporation of PI(FIG. 6). This was confirmed by immunofluorescence microscopy (FIG. 6).Cells reacted with anti-RTF and 0.75 mM ATP together were found to bepermeable to PI, but not to trypan blue (FIG. 6). In contrast, controlcells permeabilized with 70% ETOH were positive for both dyes (data notshown). Cells that were incubated with anti-RTF alone or 0.75 mM ATPalone (FIG. 6) showed no PI incorporation, while those incubated withboth did incorporate PI. These results were confirmed using luciferyellow (not shown). This demonstrates that anti-RTF and ATP together caninduce the opening of P2Z channels >0.9 kDa, and yet shows membraneintegrity is still intact.

Example 12 RTF Regulates IL-1β Secretion

[0044] To show that RTF regulates IL-1β secretion, THP-1 macrophageswere incubated with both 1 mM and 3 mM ATP, or anti-RTF and assayed forIL-1β secretion by ELISA. Since anti-RTF abrogates RTF function, ourhypothesis was that anti-RTF would induce IL-β secretion, showing thatRTF is a regulator for this molecule.

[0045] 5×10⁵ THP macrophage cells were stimulated with 5 ng/ml of LPSand 1×10⁵ PBMC for 2.5 hours. These cells were then washed and incubatedin a final concentration of either 0 ATP, 3 mM ATP, 1 mM ATP, oranti-RTF for a period of 3 hours. The supernatants were assayed forIL-1β by ELISA (R&D systems). pg IL-1β per well was calculated from theoptical densities. Results are expressed as the mean of 3 independentexperiments.

[0046] As shown in FIG. 7, cells incubated with media alone, secreted61+/−14 pg IL-1β, forming the baseline. Cells incubated with 3 mM ATPsecreted 285+/−25 pg (78+/−5.1% above baseline). Cells incubated with 1mM ATP secreted 130+/−44 pg IL-1β (49+/−5.6 above baseline). Cellsincubated with anti-RTF alone secreted 114+/−22 pg IL-1β (an increase47+/−3.5%), about the equivalent of cells incubated with 1 mM ATP. Thisshows that anti-RTF alone can induce secretion, suggesting that RTF is aregulator of IL-10.

Example 13 Anti-RTF is Cytotoxic to Ovarian Carcinoma Cell

[0047] A2780 ovarian carcinoma cells were grown in 96 well plates until100% confluent. Wells were identified, and cells were then incubatedwith 3 mM ATP, 6 mM ATP, 20 μg of anti-RTF and 20 ug of anti-RTFcombined with 3 mM ATP and 6 mM ATP. Cells were incubated for 18 hoursat 37° C. in a 5% CO₂ environment. Cells were then harvested and assayedusing flow cytometry for Caspase 3 activation as an indicator for celldeath. As shown in FIG. 8, anti-RTF is significantly more cytotoxic thenextracellular ATP at the indicated concentrations. Addition ofextracellular ATP did not significantly enhance cytotoxicity.

Example 14 In Vivo Effect of Anti-RTF

[0048] Female athymic nude mice purchased from Harlan Sprague Dawleyapproximately 11 weeks old. All mice were injected with 200 μl of a 1:1slurry of a Matrigel mix containing 5×10⁶ A2780 ovarian carcinoma cells.Tumor cells were transplanted subcutaneously in the upper flank of theright leg. Two mice were untreated and kept as a control to measuretumor growth. The remaining six mice were injected with anti RTFantibody. All mice were left untreated for 1 week in order for a tumorand its vascular bed to become established. On the eighth day all micethat were treated were injected with 100 μg of anti RTF antibody in avolume of 100 μl of PBS. Injections were given in the tumor bearingflank of the animal. Antibody was not injected directly into the tumor,but was injected into the area of the tumor bed. Mice received 300 μg ofantibody per week using a 28 gage needle. Mice were injected everySunday, Tuesday and Friday. Injections took place over 5 weeks. Theexperiment was terminated when the control mice tumor burden became tooobstructive for mice to ambulate.

[0049] The median tumor volume of the control animals after 5 weekswithout anti-RTF treatment was 8448 mm³. The median tumor volume for theanti-RTF treated animals (n=6) was 364 mm³. In two of the treatedanimals no measurable tumor was observed (tumor volume range 0-1670).These results clearly indicate the possible usefulness of the antibodyand or the inhibition of the RTF associated ATPase complex in tumortherapy

[0050] It should be understood that various changes and modifications tothe presently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

The invention is claimed as follows:
 1. A method of modulatinginflammatory and immune responses in a subject in need thereof, thesubject having a first level of regeneration and tolerance factor (RTF)activity, and the method comprising altering the first level of RTFactivity to a second level of RTF activity.
 2. The method of claim 1,wherein the modulation of the immune response is immune activation andwherein the second level of RTF activity is lower than the first levelof RTF activity.
 3. The method of claim 2, wherein the method ofenhancing the immune response is by inhibiting RTF activity.
 4. Themethod of claim 3, wherein the method of inhibiting RTF activity is byadministering to the subject an effective amount of a RTF antagonist. 5.The method of claim 4, wherein the RTF antagonist is selected from thegroup consisting of small molecules, peptides, and antibodies or afragment thereof, wherein the RTF antagonist inhibits RTF by binding toRTF.
 6. The method of claim 5, wherein the antibody is selected from thegroup consisting of monoclonal antibodies and polyclonal antibodies. 7.The method of claim 4, wherein the RTF antagonist inhibits cellularexpression or synthesis of RTF.
 8. The method of claim 7, wherein theRTF antagonist inhibits cellular RTF expression or synthesis byinterfering with DNA encoding the RTF.
 9. The method of claim 8, whereinthe RTF antagonist is an antisense nucleic acid.
 10. The method of claim7, wherein the RTF antagonist inhibits cellular RTF expression orsynthesis by inducing interference of RTF gene RNA.
 11. The method ofclaim 10, wherein the RTF antagonist is an interference RNA.
 12. Themethod of claim 11, wherein the interference RNA is a small interferenceRNA (siRNA).
 13. The method of claim 11, wherein the double-stranded RNAis exogenous.
 14. The method of claim 11, wherein the double-strandedRNA is expressed intracellularly.
 15. The method of claim 2, wherein theactivation of the immune response leads to apotosis of cancer cells. 16.The method of claim 15, wherein the cancer cell is ovarian carcinomacell.
 17. The method of claim 1, wherein the modulation of inflammatoryresponses is inhibiting inflammation wherein the second level of RTFactivity is higher than the first level of RTF activity.
 18. The methodof claim 17, wherein the first level of RTF activity is increased to thesecond level of RTF activity is by inducing cellular expression of RTF.19. The method of claim 17, wherein the first level of RTF activity isincreased to the second higher level of RTF activity is by administeringto the subject an effective amount of a RTF or a fragment thereof. 20.The method of claim 19, wherein the administered RTF or its fragment isisolated or purified from a mammalian cell.
 21. The method of claim 20,wherein the mammalian cell is a T-lymphocyte.
 22. The method of claim21, wherein the T-lymphocyte is an activated T-lymphocyte.
 23. Themethod of claim 20, wherein the mammalian tissue is a B-lymphocyte. 24.The method of claim 23, wherein the B-lymphocyte is an activatedB-lymphocyte.
 25. The method of claim 20, wherein the mammalian cell isa macrophage.
 26. The method of claim 20, wherein the mammalian cell isa thymus cell.
 27. The method of claim 20, wherein the mammalian cell isfrom fetalplacental tissue.
 28. The method of claim 20, wherein themammalian cell is a murine cell.
 29. The method of claim 20, wherein themammalian cell is a human cell.
 30. The method of claim 19, wherein theadministered RTF is an intracellular form having a molecular weight ofabout 70 kDa.
 31. The method of claim 19, wherein the administered RTFis an external membrane form having a molecular weight of about 50 kDa.32. The method of claim 19, wherein the administered RTF or its fragmentis synthetic or produced by a recombinant process.
 33. The method ofclaim 32, wherein the administered RTF or the fragment is synthesizedfrom a nucleic acid sequence encoding an amino acid sequence of the RTF.34. The method of claim 1, wherein the subject is a mammal.
 35. Themethod of claim 1, wherein the subject is a human.
 36. A method oftreating cancer in a subject in need thereof, the subject having a firstlevel of regeneration and tolerance factor (RTF) activity, and themethod comprising altering the first level of RTF activity to a secondlevel of RTF activity wherein the second level of RTF activity is lowerthan the first level of RTF activity.
 37. The method of claim 36,wherein the method of lowering the first level of RTF activity to thesecond level of RTF activity is by inhibiting the level of RTF activityin the subject.
 38. The method of claim 36, wherein the method ofinhibiting the RTF activity is by administering to the subject aneffective amount of a RTF antagonist.
 39. The method of claim 38,wherein the RTF antagonist is selected from the group consisting ofsmall molecules, peptides, and antibodies or fragments thereof, whereinthe RTF antagonist binds to RTF.
 40. The method of claim 39, wherein theantibody is selected from the group consisting of monoclonal antibodiesand polyclonal antibodies.
 41. The method of claim 38, wherein the RTFantagonist inhibits cellular expression or synthesis of RTF.
 42. Themethod of claim 41, wherein the RTF antagonist inhibits cellular RTFexpression or synthesis by interfering with DNA encoding the RTF. 43.The method of claim 42, wherein the RTF antagonist is an antisensenucleic acid.
 44. The method of claim 41, wherein the RTF antagonistinhibits cellular RTF expression or synthesis by inducing interferenceof RTF gene RNA.
 45. The method of claim 44, wherein the RTF antagonistis an interference RNA.
 46. The method of claim 45, wherein theinterference RNA is a small interference RNA (siRNA).
 47. The method ofclaim 45, wherein the double-stranded RNA is exogenous.
 48. The methodof claim 45, wherein the double-stranded RNA is expressedintracellularly.
 49. The method of claim 36, wherein the subject is amammal.
 50. The method of claim 36, wherein the subject is a human. 51.The method of claim 36, wherein the cancer is ovarian cancer.
 52. Amethod of treating or ameliorating an inflammatory disorder or anautoimmune disorder or one or more symptoms thereof, the methodcomprising administering to a subject in need thereof a prophylacticallyor therapeutically effective amount of a regeneration and tolerancefactor (RTF) or a fragment thereof.
 53. The method of claim 52, whereinthe RTF or the fragment is isolated or purified from a mammalian cell.54. The method of claim 53, wherein the mammalian cell is aT-lymphocyte.
 55. The method of claim 54, wherein the T-lymphocyte is anactivated T-lymphocyte.
 56. The method of claim 53, wherein themammalian cell is a B-lymphocyte.
 57. The method of claim 56, whereinthe B-lymphocyte is an activated T-lymphocyte.
 58. The method of claim53, wherein the mammalian cell is a macrophage.
 59. The method of claim53, wherein the mammalian cell is a thymus cell.
 60. The method of claim53, wherein the mammalian cell is from fetalplacental tissue.
 61. Themethod of claim 53, wherein the mammalian cell is a murine cell.
 62. Themethod of claim 53, wherein the mammalian cell is a human cell.
 63. Themethod of claim 52, wherein the RTF is an intracellular form having amolecular weight of about 70 kDa.
 64. The method of claim 52, whereinthe RTF is an external membrane form having a molecular weight of about50 kDa.
 65. The method of claim 52, wherein the RTF or the fragment issynthetic or prepared by a recombinant process.
 66. The method of claim65, wherein the RTF or the fragment is synthesized from a nucleic acidsequence encoding an amino acid sequence of the RTF.
 67. The method ofclaim 52, wherein the RTF or the fragment is formulated with one or moreacceptable pharmaceutical excipients.
 68. The method of claim 67,wherein the RTF or the fragment is formulated as nanoparticles ormicroparticles.
 69. The method of claim 68, wherein the nanoparticles ormicroparticles further comprising ligands for delivery of thenanoparticles or microparticles to a target tissue.
 70. The method ofclaim 69, wherein the target is a T-lymphocyte.
 71. The method of claim69, wherein the target is a B-lymphocyte.
 72. The method of claim 69,wherein the target is a macrophage.
 73. The method of claim 67, whereinthe RTF or the fragment is administered by a route selected from thegroup consisting of: parenteral, topical, nasal, pulmonary, ophthalmic,bucal, vaginal, transdermal, intrathecal, and oral.
 74. The method ofclaim 52, wherein the subject is a mammal.
 75. The method of claim 52,wherein the subject is human.
 76. The method of claim 52, wherein theinflammatory disorder is arthritis.
 77. The method of claim 52, furthercomprising the step of administering to the subject a TNF-α antagonists.78. The method of claim 77, wherein the TNF-α antagonists is infliximab.79. The method of claim 77, wherein the TNF-α antagonist is etanercept.80. The method of claim 77, wherein the TNF-α antagonist is D2E7. 81.The method of claim 77, wherein the TNF-α antagonist is CDP571.
 82. Themethod of claim 77, wherein the TNF-α antagonist is CDP870.
 83. Themethod of claim 77, wherein the TNF-α antagonist is a thalidomideanalog.
 84. The method of claim 77, wherein the TNF-α antagonist is aphosphodiesterase type IV inhibitor.
 85. A composition for enhancingimmune response in a subject in need thereof, the composition comprisingan effective amount of a RTF antagonist.
 86. The composition of claim85, wherein the RTF antagonist is selected from the group consisting ofsmall molecules, peptides, antisense nucleic acids, interference RNAs,and antibodies or fragments thereof.
 87. The composition of claim 86,wherein the antibody is polyclonal or monoclonal.
 88. The composition ofclaim 86, wherein the antibody is monoclonal.
 89. The composition ofclaim 86, wherein the interference RNA is small interference RNA(siRNA).
 90. The composition of claim 85 further comprising a suitablepharmaceutical excipient.
 91. The composition of claim 85, wherein thecomposition is suitable for administering to the subject by a routeselected from the group consisting of: parenteral, topical, nasal,pulmonary, ophthalmic, bucal, vaginal, transdermal, intrathecal, andoral.
 92. The composition of claim 85, wherein the composition isformulated as nanoparticles or microparticles.
 93. The composition ofclaim 86, wherein enhancing immune response leads to apotosis of cancercells.
 94. The composition of claim 93, wherein the cancer cell isovarian carcinoma cancer cell.
 95. The composition of claim 85, whereinthe subject is a mammal.
 96. The composition of claim 85, wherein thesubject is a human.
 97. A composition for enhancing treating cancer in asubject in need thereof, the composition comprising an effective amountof a RTF antagonist.
 98. A composition for treating or ameliorating aninflammatory disorder or an autoimmune disorder or one or more symptomsthereof in a subject, the composition comprising an effect amount of aregeneration and tolerance factor (RTF) or a fragment thereof.
 99. Thecomposition of claim 98, wherein the RTF or the fragment is isolated orpurified from a mammalian cell.
 100. The composition of claim 99,wherein the mammalian cell is a T-lymphocyte.
 101. The composition ofclaim 100, wherein the T-lymphocyte is an activated T-lymphocyte. 102.The composition of claim 99, wherein the mammalian cell is aB-lymphocyte.
 103. The composition of claim 102, wherein theB-lymphocyte is an activated T-lymphocyte.
 104. The composition of claim99, wherein the mammalian cell is a macrophage.
 105. The composition ofclaim 99, wherein the mammalian cell is a thymus cell.
 106. Thecomposition of claim 99, wherein the mammalian cell is fromfetalplacental tissue.
 107. The composition of claim 99, wherein themammalian cell is a murine cell.
 108. The composition of claim 99,wherein the mammalian cell is a human cell.
 109. The composition ofclaim 98, wherein the RTF is an intracellular form having a molecularweight of about 70 kDa.
 110. The composition of claim 98, wherein theRTF is an external membrane form having a molecular weight of about 50kDa.
 111. The composition of claim 98, wherein the RTF or the fragmentis synthetic or prepared by a recombinant process.
 112. The compositionof claim 111, wherein the RTF or the fragment is synthesized from anucleic acid sequence encoding an amino acid sequence of the RTF. 113.The composition of claim 98, wherein the RTF or the fragment isformulated with one or more acceptable pharmaceutical excipients. 114.The composition of claim 98, wherein the RTF or the fragment isformulated as nanoparticles or microparticles.
 115. The composition ofclaim 114, wherein the nanoparticles or microparticles furthercomprising ligands for delivery of the nanoparticles or microparticlesto a target tissue.
 116. The composition of claim 115, wherein thetarget is a T-lymphocyte.
 117. The composition of claim 115, wherein thetarget is a B-lymphocyte.
 118. The composition of claim 115, wherein thetarget is a macrophage.
 119. The composition of claim 98, wherein theRTF or the fragment is administered by a route selected from the groupconsisting of: parenteral, topical, nasal, pulmonary, ophthalmic, bucal,vaginal, transdermal, intrathecal, and oral.
 120. The composition ofclaim 98, wherein the subject is a mammal.
 121. The composition of claim98, wherein the subject is human.
 122. The composition of claim 98,wherein the inflammatory disorder is arthritis.